Field of the Invention
The present invention relates to a method for generating a cancer stem cell line from an immortalized cell line. More particularly, the present invention relates to a method for generating a novel cancer stem cell line that possesses characteristics associated with stem cells, by co-culturing a human immortalized cell line and bone marrow-derived mesenchymal stem cells, and the novel cancer stem cell line established thereby.
Description of the Related Art
Cancer stem cells (hereinafter referred to as “CSCs”, interchangeably used for tumor stem cells) are primary tumor cells that exhibit self-renewal, proliferative capacity and multi-differentiation, forming a microenvironment where blood vessels, mesenchymal stem cells and a variety of cancer cells are found. Now, there are four methods of isolating cancer stem cells from tumor tissue using characteristics of cancer stem cells:
first, MACS (Magnetic-Activated Cell Sorting) and FACS (Fluorescence-Activated Cell Sorting), using antibodies against specific markers expressed by tumor stem cells;
second, side population discrimination by a dye exclusion assay that takes advantage of the high activity of an ABC reporter predominantly in leukemia and multiple myeloma;
third, isolation using the activity of such enzymes that are overexpressed in tumor stem cells as aldehyde dehydrogenase (ALDH); and
fourth, segregation of tumor stem cells from differentiated cancer cells by forming a tumorsphere in the presence of growth factors (EGF and bFGF) in a serum-free medium.
These methods, although readily isolating CSCs from cancer cells, are not effective in securing a multitude of CSCs since the CSCs are different in ratio from each other. Also, the CSCs isolated by each isolation method differ in oncogenesis from one to another. In addition, since characteristics of CSCs are changed during cultivation, isolation and culture methods are further studied. Moreover, it takes a significant time to obtain tumor tissues from cancer patients according to legal and administrative matters.
Hence, there is a need for a method for generating cancer stem cells that overcomes the conventional problem of isolating a small population of cancer stem cells and that can secure a multitude of homogenous cancer stem cells, with few legal or administrative limitations.
As a background of the present invention, Korean Patent No. 10-1242726 (issued Mar. 13, 2013) discloses a method of diagnosing cancer with a cancer stem cell property by measuring the expression level of aldehyde dehydrogenase (ALDH). Korean Patent Publication No. 10-2012-0099904 (issued Sep. 12, 2012) describes a tumor stem cell-targeting peptide GICT that can selectively recognize and bind malignant, undifferentiated glioma cells, and a method for diagnosing encephaloma. Korean Patent No. 10-0783199 (issued Dec. 6, 2007) describes a cancer stem cell line GBM 2, established from human glioblastoma multiforme tissue, having resistance to drugs.
However, nowhere is a method of easily, quickly and efficiently generating cancer stem cells from an immortalized cell line and maintaining the cancer stem cells found in previous literature.
Leading to the present invention, intensive and thorough research into the mass production of cancer stem cells having high oncogenic potential resulted in the finding that cancer stem cells can be derived from an immortalized cell line which is co-cultured with mesenchymal stem cells.
References relevant to the subject matter of the present disclosure include the following:
In vitro generation of human cells with cancer stem cell properties. Scaffidi P, Misteli T. Nat Cell Biol. 2011 Aug. 21; 13(9):1051-61.
SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Son M J, Woolard K, Nam D H, Lee J, Fine H A. Cell Stem Cell. 2009 may 8; 4(5):440-52
CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors. Patru C, Romao L, Varlet P, Coulombel L, Raponi E, et al., BMC Cancer. 2010 Feb. 24; 10:66.
The micro-RNA 199b-5p regulatory circuit involves Hest, CD15, and epigenetic modifications in medulloblastoma. Andolfo I, Liguori L, De Antonellis P, Cusanelli E, Marinaro F, Pistollato F, Garzia L, et al., Neuro-Oncology. 2012 Jan. 22; 14(5):596-612.